Oxygen free radicals are believed to be among the major possible causative agents of a number of pathological conditions such as cancer, aging, arthritis, emphysema and infant retinopathy among others. One mode of action of oxygen-derived species is to cause DNA lesions, resulting in cell mutation or death. The goal of this study is to quantitatively establish the extent to which oxygen free radicals are responsible for in vivo DNA damage in cultured mammalian cells. A model murine hybridoma system producing monoclonal IgG antibodies has been chosen for our studies. In order to attain our objective, the following specific questions will be answered sequentially: i) Can oxidative DNA lesions be quantitatively measured in chromatin? Contemporary measurements of chemical modifications to DNA have been made on isolated DNA, which do not portray a realistic picture of actual damage occurring at the chromatin level. This is because DNA present in chromatin is structurally sequestered and bound to histones. Furthermore, present state-of-the-art measurements of DNA damage in mammalian cells consist primarily of identifying strand breakage. There is a need for a more powerful and quantitative assay. A superior approach is available through the use of Gas Chromatography/Mass Spectrometry techniques to precisely and quantitatively identify DNA lesions. Several base products have been identified through this technique by exposing isolated DNA to free radical attack. It is proposed to expose isolated intact chromatin to free radical attack under controlled conditions and subsequently characterize the damage. ii) Are the same lesions found in cells in vivo? This question has not been addressed to any significant degree in mammalian cells. We propose to use in vitro bioreactor culture techniques, which allow for the precise control of all environmental and physiological factors. Live cells in controlled environments will be exposed (as in the case of isolated chromatin) to known free radical generating systems. Based on the methodology developed to answer the first question, this approach will permit accurate studies of in vivo DNA damage in cultured mammalian cells. Additionally, it will be possible to study the kinetics of DNA repair by monitoring the temporal disappearance of damaged bases from cellular chromatin after exposing cells to free radicals. In addition to having a major impact on contemporary research in the field of oxygen toxicity, this work will assist in meeting the objectives of the RFA by not only developing hybridomas as a high connectivity mammalian system but also in promoting the use of in vitro tissue culture experiments as an alternative to animal use. In the longer term, the studies proposed here will lay the groundwork for understanding conditions and mechanisms by which oxidative DNA damage and subsequent repair occurs in mammalian cells.